EP0140764B1 - Verfahren und Reagenzien zum Nachweis von normalerweise einkettigen Nukleinsäuren, insbesondere RNS, in einem biologischen Material, insbesondere in einem Präparat von Leukocyten - Google Patents

Verfahren und Reagenzien zum Nachweis von normalerweise einkettigen Nukleinsäuren, insbesondere RNS, in einem biologischen Material, insbesondere in einem Präparat von Leukocyten Download PDF

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EP0140764B1
EP0140764B1 EP84401974A EP84401974A EP0140764B1 EP 0140764 B1 EP0140764 B1 EP 0140764B1 EP 84401974 A EP84401974 A EP 84401974A EP 84401974 A EP84401974 A EP 84401974A EP 0140764 B1 EP0140764 B1 EP 0140764B1
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rna
monocatenary
determined
nucleic acid
fact
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EP0140764A1 (de
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Michel Crepin
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Institut Pasteur de Lille
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Institut Pasteur de Lille
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/803—Physical recovery methods, e.g. chromatography, grinding
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/81—Packaged device or kit
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function
    • Y10S436/813—Cancer
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/825—Pretreatment for removal of interfering factors from sample
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/25—Chemistry: analytical and immunological testing including sample preparation
    • Y10T436/25125—Digestion or removing interfering materials

Definitions

  • the invention relates to a method and reagents for the detection of a genetic abnormality in a complex biological material, for example a tissue or, preferably, a preparation of blood cells, such as leukocytes. It relates more particularly to the application of this method to in vitro detections of genetic abnormalities involving in particular defective genes of oncogenes or exogenous genes capable of being incorporated into the tissues, leukocytes or other cells of the organism and of express themselves there.
  • a complex biological material for example a tissue or, preferably, a preparation of blood cells, such as leukocytes.
  • mRNA messenger RNA
  • the mRNA may reflect either an alteration of the genetic heritage of the host, or an induction of the expression of genes present in the organism, but normally not expressed, either an invasion or an infiltration of the organism or the host from which the material originates by foreign constituents, carrying genes capable of being themselves expressed in this organism.
  • Examples of genetic abnormalities include the often altered oncogenes, which are expressed in the corresponding tumors.
  • oncogenes A number of genes involved in oncogenesis have already been described in the literature (Myb, Myc, Erb ).
  • Myc gene is activated in myelocytomatosis.
  • the invention therefore aims to promote a detection method which can account for the earliest stages of the development of the desired disease. She was therefore particularly interested in the first sequences of the series of events which, on the biological level, lead to the expression products characteristic of the affection or anomaly studied. One of these early events was the transcription into mRNA of the genetic defect that caused it.
  • the invention also aims to provide a method making it possible to carry out detections of the presence or not of an RNA within a biological sample, which is both more sensitive and more simple to implement than conventional methods for detecting a particular RNA.
  • the invention aims to remedy the difficulties involved in the implementation of conventional methods, these generally involving extensive purifications of the RNAs before contacting them with a probe capable of hybridize with the particular RNA sought.
  • the invention therefore also aims to significantly reduce the preliminary treatments necessary for obtaining an RNA preparation in a suitable state for the detection step, in particular by hybridization with a probe marked, can actually be implemented. It therefore further aims to reduce the loss of RNA to detect that entail the usual purification procedures.
  • the invention applies with a particular advantage to the detection of mRNAs involved in the expression of particular genes, as soon as suitable probes are available.
  • a first element on which the invention is based is that the detection of a messenger RNA - or more generally of any RNA possibly present - in a biological sample is made possible as soon as this RNA can be "unmasked", this unmasking implying an at least partial dissociation of the complexes that these RNAs appear to form with other cellular constituents, in particular proteins. It is then not necessary to carry out the complete purification of the RNA sought.
  • the second element on which the method according to the invention is based resides in the gentle treatment, as defined above.
  • the non-denaturation, in any case in significant proportions, of double-stranded nucleic acids reinforces the selectivity of the subsequent hybridization operation of the RNA sought with the labeled probe. Double-stranded DNA cannot in fact intervene with the hybridization reaction.
  • RNA detection method according to the invention is therefore particularly advantageous because of its simplicity and its sensitivity, the latter being further increased by limiting the loss of the particular RNA sought to a minimum.
  • the method according to the invention is applied to the detection of an RNA, in particular of a messenger RNA corresponding to a DNA sequence affected by a genetic abnormality.
  • the invention is also applicable to the detection of any single-stranded nucleotide sequence foreign to the biological material studied.
  • the invention can be applied to the detection of expression products of viruses, for example of the hepatitis B virus, or of retroviruses, the presence of which can attest to the development of a specific condition in humans. host from which the biological sample comes.
  • viruses for example of the hepatitis B virus, or of retroviruses
  • the invention can be applied to the detection of expression products of viruses, for example of the hepatitis B virus, or of retroviruses, the presence of which can attest to the development of a specific condition in humans. host from which the biological sample comes.
  • lymphadenopathies or acquired immune deficiency syndrome thanks to the detection of the retrovirus which appears to be associated therewith (F. Barré- Sinoussi and Coll. Science, May 20, 1983, 220, 868 - 871).
  • the invention finds a particularly important application in the field of early in vitro diagnosis of malignant tumors or leukemias. It leads to particularly significant results whenever an oncogenic gene (hereinafter often referred to as "oncogene") linked to the type of tumor in question has been identified. In such a case, detection will be based on carrying out a hybridization between the corresponding mRNA, the production of which constitutes the first stage of the expression of the corresponding gene, with a probe containing a nucleotide sequence corresponding to this gene.
  • oncogene oncogenic gene
  • the method of the invention is applicable to the early detection of other tumors, in particular primary solid tumors, even when specific oncogenes linked to this type of tumor have not yet been isolated. It has in fact been observed that the development within an organism of a given tumor is often accompanied by an induction of the expression of oncogenes, apparently not directly involved in the tumor in question. This is what is observed in the case of human mammary carcinomas, which can be detected in vitro by implementing the method according to the invention with a probe hybridizable with the Myc gene, with a degree of certainty at least equivalent to that of the results provided by other methods used in this field (with the exception of anatomo-pathological sections).
  • RNA to be detected is not necessarily directly linked to the very biological cause of the condition which must be diagnosed. It suffices that there can be a correlation between the RNA to be detected and the condition to be diagnosed.
  • the biological material of choice for implementing the method according to the invention consists of a preparation of blood cells, preferably leukocytes.
  • the observations which have been made support the hypothesis that a proliferation of tumor cells in the organism can in many cases be accompanied by sufficient induction of a large variety of the oncogenes present, so that their expression is detectable by the method according to the invention, using a probe complementary to one of them.
  • the invention is therefore of particularly important application for specifying a diagnosis and a prognosis on the development of neoplasias giving rise to metastases (pulmonary, ganglionic ...), in that the degree infiltration of the organism, and in particular of the blood by lymphoblastoid cells, appears to be directly linked to the malignancy of the tumor.
  • the gentle treatment of the cells to be treated comprises bringing the cells of the material under study, preferably blood leukocytes, into contact with a nonionic detergent, such as those known under the trade names or not, NONIDET 40 (NP 40), BRIJ 35, deoxycholate, etc. and bringing those of the released cellular constituents, likely to contain the messenger RNAs, into contact with a solution of an ionic salt of high molarity, for example a concentrated solution of potassium iodide or of sodium to both dissolve and dissociate the complexes formed by RNAs with proteins or other constituents.
  • a nonionic detergent such as those known under the trade names or not, NONIDET 40 (NP 40), BRIJ 35, deoxycholate, etc.
  • a solution of an ionic salt of high molarity for example a concentrated solution of potassium iodide or of sodium to both dissolve and dissociate the complexes formed by RNAs with proteins or other constituents.
  • Cell extracts can then be subjected to the RNA detection procedure complementary to that of the chosen probe. Any known technique can be used in this regard. It is advantageous to use in situ nitrocellulose hybridization techniques. For this purpose, a small volume of the solution of the cell extract and the ionic salt is deposited on the cellulose filter. The latter is then washed with the solutions making it possible to remove the excess ionic salt, proteins and the probe, which is not attached. It is advantageous to carry out these washes with water, solutions of an ionic detergent such as sodium dodecyl sulfate, and ethanol. The latter also contributes to fixing the precipitated RNA on the filter and facilitates subsequent detection, by bringing the filter into contact with a labeled probe.
  • an ionic detergent such as sodium dodecyl sulfate
  • any conventional technique can be used for labeling the probe.
  • the latter is radioactively labeled or coupled (or couplable) to an enzyme which can be revealed by the action which it is capable of exerting with regard to a determined substrate, or to a fluorescent molecule.
  • this kit also includes the means for carrying out an in situ hybridization between the probe (s) and the complementary RNA possibly present in the biological medium studied.
  • the kit then comprises a filter or the like on which the RNAs can be deposited, and a reagent for fixing the messenger RNA. on the filter, such as ethanol, reagents for the removal by washing of proteins, lipids and other residual cellular constituents.
  • This kit advantageously further comprises a reagent such as acetic anhydride in triethanolamine, capable of preventing the attachment of the probe to the filter, via bonds other than those resulting from hybridization.
  • a reagent such as acetic anhydride in triethanolamine
  • a non-limiting example indicates an advantageous procedure for carrying out a standard test for detecting the expression of an oncogene in a biological sample from a determined patient.
  • a blood sample is subjected to treatment to lyse red blood cells, for example by contacting with an ammonium chloride solution.
  • the sample is centrifuged at low speed, for example at 1000 rpm for 5 minutes.
  • the centrifugation pellet containing the leukocytes is recovered.
  • the leukocytes collected are washed twice in a phosphate buffer (PBS) and dissociated in a mixture of two detergents at 0.5% (NP 40 and deoxycholate). After an incubation at 0 ° C for a period of 5 to 15 minutes depending on the nature of the tissue, the cells are treated at ordinary temperature with sodium iodide (Nal, 6.1 M).
  • the cell extract obtained can be diluted in a saturated solution of Nal (12.2 M) and deposited in a small volume (a few microliters) on a nitrocellulose filter (Schleischer and Schull).
  • the filters are washed for 5 minutes in water, then in a 1% sodium dodecyl sulfate solution. Then three washes of 5 minutes are carried out in 70% ethanol to remove the sodium iodide. Finally, washing for 10 minutes in 20 ml of a solution containing 0.25 ml of acetic anhydride in 100 ml of triethanolamine will eliminate the background noise from the test.
  • the filters are dried and can be stored in a desiccator.
  • the filter is then treated and hybridized overnight with labeled DNA probe (containing for example a sequence hybridizable with one of the oncogenes Myc, Myb, Erb, etc.), for example as described by Wahl et al. (1979) ...
  • labeled DNA probe containing for example a sequence hybridizable with one of the oncogenes Myc, Myb, Erb, etc.
  • the hybridized homologous sequences (Myc, Myb, Erb) can, for example, be detected by autoradiography of the radioactive probe or with an antibiotic antiserum recognizing the probe DNA, when a probe is used. to which biotin has been coupled (or can be coupled).
  • the intensity of the spot makes it possible to precisely assess the degree of expression of the oncogene per cell, per gram of tissue or per milliliter of blood.
  • the filter always containing an internal standard can be rehybridized with another labeled probe.

Claims (19)

1. Verfahren zum in vitro Nachweis einer normalerweise einkettigen Nukleinsäure, insbesondere einer bestimmten RNS, in einem sie und Zellen enthaltenden biologischen Material, mit den folgenden Verfahrenschritten:
- die Behandlung des biologischen Materials durch die Herstellung eines Kontakts des Materials mit einem nicht ionischen Detergens, um die Membranen der in dem biologischen Material enthaltenen Zellen lösbar zu machen und einen Teil der internen Zellenbestandteile freizusetzen, darunter DNS, RNS und insbesondere die Messenger-Ribonkleinsäuren,
- die Dissoziierung vor der Hybridisierung der Komplexe von Protein und einkettiger RNS und Demaskierung der einkettigen RNS mit Hilfe einer Lösung eines ionischen Salzes der Molarität 5 - 13 M, wobei diese schonende Behandlung im wesentlichen die Denaturierung der doppelkettigen Nukleinsäuren vermeidet,
- das Inkontaktbringen der RNS mit einer eine komplementäre Nukleotidsequenz der bestimmten einkettigen Nukleinsäure, insbesondere der bestimmten RNS, enthaltenden Sonde unter Bedingungen, die geeignet sind, die Hybridisierung dieser Sonde mit dieser bestimmten Nukleinsäure zu erlauben,
- die Hybridisierung zwischen der genannten Sonde und der bestimmten Sequenz der einkettigen Nukleinsäure, insbesondere der einkettigen RNS, in Anwesenheit von im wesentlichen doppelkettigen Nukleinsäuren,
- die Feststellung des gegebenenfalls gebildeten Hybrids zwischen der bestimmten Nukleinsäure und der komplementären Sequenz der Sonde,
- dadurch gekennzeichnet, daß man die RNS auf ein für die Hybridisierung in situ geeignetes Filter aufbringt und mit einem ionischen Detergenz wäscht, um die Proteine zu eliminieren.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daB man die RNS auf ein für die Hybridisierung in situ geeignetes Filter bringt und mit einem ionischen Detergenz wäscht, um die Proteine zu eliminieren, bevor man eine Acetylierung oder dergleichen des Filters vornimmt, um Hintergrundstörungen zu vermeiden.
3. Verfahren nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß man nach der Aufbringung der RNS auf das Filter und dem Waschen mit einem ionischen Detergens mit Essigsäureanhydrid in Triethanolamin wäscht, um Hintergrundstörungen zu eliminieren.
4. Verfahren nach den Ansprüchen 1 - 3, dadurch gekennzeichnet, daß das ionische Detergenz zum Eliminieren der Proteine durch Waschen Natriumdodecylsulfat ist.
5. Verfahren nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die nachzuweisende RNS aus einer Messenger-RNS besteht, die bei der Expression eines bestimmten Gens interveniert.
6. Verfahren nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die nachzuweisende RNS charakteristisch für eine genetische Anomalie ist.
7. Verfahren nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die nachzuweisende RNS der normalerweise einkettigen Nukleinsäure dem genetischen Erbgut der Zellen fremd ist und normalerweise von einer viralen DNS oder einer viralen RNS gebildet wird.
8. Verfahren nach einem der Ansprüche 1 - 4, dadurch gekennzeichnet, daß die bestimmte RNS diejenige ist, die aus der Transkription eines Onkogens resultiert.
9. Verfahren nach einem der Ansprüche 1 - 8, dadurch gekennzeichnet, daß das biologische Muster von Zellenbestandteilen des Blutes gebildet wird.
10. Verfahren nach einem Anspruch 9, dadurch gekennzeichnet, daß das biologische Muster eines auf Basis von Leukozyten ist.
11. Anwendung des Verfahrens nach einem der Ansprüche 1 - 10 zum in vitro Nachweis einer genetischen Anomalie mit pathologischem Charakter durch das Inkontaktbringen der oben genannten zellulären Bestandteile mit einer Sonde, die eine Nukleotidsequenz enthält, die mit einer dem durch die genetische Anomalie betroffenen Gen entsprechenden Messenger-RNS hybridisierbar ist.
12. Anwendung des Verfahrens nach einem der Ansprüche 1 - 10 auf den in vitro Nachweis von Blastoidzellen oder von Metastasen bösartiger Tumore, gekennzeichnet durch das Inkontaktbringen der oben genannten Zellenbestandteile mit einer Komplementärsonde eines onkogenen Gens.
13. Material für die Durchführung des in vitro Nachweises einer bestimmten normalerweise einkettigen Nukleinsäure, insbesondere einer bestimmten RNS, in einem biologischen Material, enthaltend eine Anzahl von Reagentien, zu denen gehören:
- ein oder mehrere nicht-ionische Detergentien, die es unter geeigneten Verdünnungsbedingungen ermöglichen, die Membranen der in dem zu behandelnden biologischen Material enthaltenen Zellen auflösen;
- die Bestandteile und Puffer, die zur Realisierung der Lösungen der oben genannten nichtionischen Detergentien bei Konzentrationen nötig sind, die die Freisetzung mindestens eines Teils der internen Zellenbestandteile ermöglichen, unter ihnen die Messenger-Ribonkleinsäuren;
- ionische Salze, die die schonende Behandlung der Zellen des oben genannten Materials ermöglichen, u. bei einer Molarität von 5 - 13 M die Dissozierung der Komplexe Protein-RNSn, die in dem behandelten Material enthalten sind, zu realisieren und die RNSn zu demaskieren,
- die Bestandteile und Puffer, die zur Realisierung der Lösungen der oben genannten ionischen Salze bei den genannten Konzentrationen erforderlich sind, die die schonende Behandlung der Zellen des genannten Materials ermöglichen, um die Komplexe Protein-RNS zu dissozieren und die Messenger-Ribonkleinsäurenzu demaskieren,
- eine oder mehrere markierte Sonden, die eine komplementäre Nukleotidsequenz der bestimmten normalerweise einkettigen Nukleinsäure enthalten und die Mittel, die die Realisierung der folgenden Hybridisierung in situ der für die gesuchte genetische Anomalie charakteristischen RNS mit einer oder mehreren der oben genannten markierten Sonden ermöglichen, dadurch gekennzeichnet, daß es ebenfalls umfaßt:
- ein ionisches Detergenz zum Eliminieren der Proteine durch Waschen,
- die Bestandteile und Puffer, die zur Realisierung der Lösung des oben genannten ionischen Detergenz erforderlich sind.
14. Material nach Anspruch 13 dadurch gekennzeichnet, daß das biologische Material durch die Zellenbestandteile des Blutes gebildet wird.
15. Material nach Anspruch 14 dadurch gekennzeichnet, daß das biologische Material solches auf Basis von Leukozyten ist.
16. Material nach einem der Ansprüche 13 - 15 dadurch gekennzeichnet, daß die ionischen Salze Salze von Natriumjodid oder Salze von Kaliumjodid sind.
17. Material nach einem der Ansprüche 13 - 16, dadurch gekennzeichnet, daß die Mittel zur Realisierung der Hybridisierung in situ ein Filter, auf dem die Nukleinsäuren abgelegt werden können, ein Reagenz zur Fixierung der einkettigen Nukleinsäuren auf dem Filter und ein ionisches Detergens zur Eleminierung der Proteine, Lipide und anderer Restzellenbestandteile durch Waschen umfassen.
18. Material nach Anspruch 17 dadurch gekennzeichnet, daß das Fixierungsreagens Ethanol und das genannte ionische Detergens für die Eleminierung der Proteine Natriumdodecylsulfat ist.
19. Material nach einem der Ansprüche 17 oder 18, enthaltend als Reagenz Essigsäureanhydrid in Triäthanolamin, geeignet zur Verhinderung der Fixierung der Sonde auf dem Filter, über andere Bindungen als die, die sich aus der Hybridisierung ergeben.
EP84401974A 1983-10-03 1984-10-03 Verfahren und Reagenzien zum Nachweis von normalerweise einkettigen Nukleinsäuren, insbesondere RNS, in einem biologischen Material, insbesondere in einem Präparat von Leukocyten Expired - Lifetime EP0140764B1 (de)

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AT84401974T ATE51251T1 (de) 1983-10-03 1984-10-03 Verfahren und reagenzien zum nachweis von normalerweise einkettigen nukleinsaeuren, insbesondere rns, in einem biologischen material, insbesondere in einem praeparat von leukocyten.

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FR8315726 1983-10-03
FR8315726A FR2552879B1 (fr) 1983-10-03 1983-10-03 Procede et reactifs pour la detection d'une anomalie genetique dans une matiere biologique, notamment dans une preparation de leucocytes

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EP0140764A1 EP0140764A1 (de) 1985-05-08
EP0140764B1 true EP0140764B1 (de) 1990-03-21

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US (1) US5070009A (de)
EP (1) EP0140764B1 (de)
JP (1) JPH0653080B2 (de)
AT (1) ATE51251T1 (de)
DE (1) DE3481717D1 (de)
FR (1) FR2552879B1 (de)

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US5614551A (en) * 1994-01-24 1997-03-25 The Johns Hopkins University Inhibitors of fatty acid synthesis as antimicrobial agents
US6958392B2 (en) * 1998-10-09 2005-10-25 Whatman, Inc. Methods for the isolation of nucleic acids and for quantitative DNA extraction and detection for leukocyte evaluation in blood products

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FR2152477A1 (en) * 1971-09-17 1973-04-27 Merieux Inst Interferon-inducers - by extraction of cells inoculated with rna virus
US4038143A (en) * 1973-10-29 1977-07-26 The Regents Of The University Of Michigan Test kit for the genetic detection of microorganisms
FR2422956A1 (fr) * 1978-04-13 1979-11-09 Pasteur Institut Procede de detection et de caracterisation d'un acide nucleique ou d'une sequence de celui-ci, et reactif enzymatique pour la mise en oeuvre de ce procede
FR2505845A1 (fr) * 1981-05-13 1982-11-19 Choay Sa Procede de dosage de la 2',5'-oligo-adenylate-synthetase, methodes de detection d'une infection virale ou microbienne, de dosage de la concentration de l'interferon circulant ou present dans des tissus, apres administration de l'interferon, ou apres administration d'inducteurs de l'interferon et ensemble pret a l'emploi, ou kit, pour la mise en oeuvre desdits procedes de dosage et methodes de detection et de dosage
CA1180647A (en) * 1981-07-17 1985-01-08 Cavit Akin Light-emitting polynucleotide hybridization diagnostic method
FR2518755B1 (fr) * 1981-12-23 1986-04-11 Pasteur Institut Sonde contenant un acide nucleique modifie et reconnaissable par des anticorps specifiques et utilisation de cette sonde pour detecter et caracteriser une sequence d'adn homologue
US4483920A (en) * 1982-05-17 1984-11-20 Hahnemann University Immobilization of message RNA directly from cells onto filter material

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FR2552879B1 (fr) 1987-08-14
JPH0653080B2 (ja) 1994-07-20
EP0140764A1 (de) 1985-05-08
DE3481717D1 (de) 1990-04-26
ATE51251T1 (de) 1990-04-15
JPS60102200A (ja) 1985-06-06
US5070009A (en) 1991-12-03
FR2552879A1 (fr) 1985-04-05

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